Streak frozen glycerol stock on appropriate agar medium and incubate 12 - 16 hours at 37 °C.
Use single colonies to inoculate 2 culture tubes containing 5 mL of liquid medium. Incubate at 37 °C for ~16 hours (rich medium) or ~24 hours (minimal medium).
Use 50 μL of saturated culture (1/300th volume) to inoculate 50 mL BioReaction tubes containing 15 mL of liquid medium. Incubate at 37 °C with 250 RPM shaking until OD600 = 1.0 (3 - 4 hours for rich medium, 10 - 12 hours for minimal medium).
Centrifuge tubes at 4,000G for 15 mins, decant excess culture medium (into 25% bleach solution) and freeze cell pellets at -80 °C.
Cell Lysis
Begin cooling the centrifuge to 4 °C.
Thaw cell pellets on ice for 15 mins, then resuspend each pellet in pre-chilled lysis buffer. Use 1 mL of lysis buffer per ~5 mL of suspension culture.
Pipette to mix until a marked increase in sample viscosity (due to released genomic DNA) is observed.
Incubate lysate on ice for 20 mins. Swirl to mix every 5 min.
Add 1 µL of 1 M MgCl2 per 5 mL of initial culture volume to the lysate (final concentration of 1 mM).
Add 0.5 µL (125 U) of benzonase nuclease per 5 mL of initial culture volume to the lysate.
Incubate lysate at 37 °C for 20 min. Invert to mix several times every 5 mins.
Chill lysates on ice for 5 mins.
Pellet remaining debris by centrifugation at 12,000G for 20 mins at 4 °C.
GELFrEE Fractionation
Proprietary method based on tube gel electrophoresis
High cost per cartridge kit - $230
Tris-acetate gel chemistry, pH 8.1
Large amount of protein needed to run a single lane (~400 µg)